Strain-Stiffening of Agarose Gels

Strain-stiffening is one of the characteristic properties of biological hydrogels and extracellular matrices, where the stiffness increases upon increased deformation. Whereas strain-stiffening is ubiquitous in protein-based materials, it has been less observed for polysaccharide and synthetic polymer gels. Here we show that agarose, that is, a common linear polysaccharide, forms helical fibrillar bundles upon cooling from aqueous solution. The hydrogels with these semiflexible fibrils show pronounced strain-stiffening. However, to reveal strain-stiffening, suppressing wall slippage turned as untrivial. Upon exploring different sample preparation techniques and rheological architectures, the cross-hatched parallel plate geometries and in situ gelation in the rheometer successfully prevented the slippage and resolved the strain-stiffening behavior. Combining with microscopy, we conclude that strain-stiffening is due to the semiflexible nature of the agarose fibrils and their geometrical connectivity, which is below the central-force isostatic critical connectivity. The biocompatibility and the observed strain-stiffening suggest the potential of agarose hydrogels in biomedical applications.Peer reviewe

Compressive stress-mediated p38 activation required for ER alpha plus phenotype in breast cancer

Breast cancer is now globally the most frequent cancer and leading cause of women's death. Two thirds of breast cancers express the luminal estrogen receptor-positive (ER alpha + ) phenotype that is initially responsive to antihormonal therapies, but drug resistance emerges. A major barrier to the understanding of the ER alpha-pathway biology and therapeutic discoveries is the restricted repertoire of luminal ER alpha + breast cancer models. The ER alpha + phenotype is not stable in cultured cells for reasons not fully understood. We examine 400 patient-derived breast epithelial and breast cancer explant cultures (PDECs) grown in various three-dimensional matrix scaffolds, finding that ER alpha is primarily regulated by the matrix stiffness. Matrix stiffness upregulates the ER alpha signaling via stress-mediated p38 activation and H3K27me3-mediated epigenetic regulation. The finding that the matrix stiffness is a central cue to the ER alpha phenotype reveals a mechanobiological component in breast tissue hormonal signaling and enables the development of novel therapeutic interventions. Subject terms: ER-positive (ER + ), breast cancer, ex vivo model, preclinical model, PDEC, stiffness, p38 SAPK. Reliable luminal estrogen receptor (ER alpha+) breast cancer models are limited. Here, the authors use patient derived breast epithelial and breast cancer explant cultures grown in several extracellular matrix scaffolds and show that ER alpha expression is regulated by matrix stiffness via stress-mediated p38 activation and H3K27me3-mediated epigenetic regulation.Peer reviewe

Compressive stress-mediated p38 activation required for ERα + phenotype in breast cancer

Breast cancer is now globally the most frequent cancer and leading cause of women's death. Two thirds of breast cancers express the luminal estrogen receptor-positive (ER alpha + ) phenotype that is initially responsive to antihormonal therapies, but drug resistance emerges. A major barrier to the understanding of the ER alpha-pathway biology and therapeutic discoveries is the restricted repertoire of luminal ER alpha + breast cancer models. The ER alpha + phenotype is not stable in cultured cells for reasons not fully understood. We examine 400 patient-derived breast epithelial and breast cancer explant cultures (PDECs) grown in various three-dimensional matrix scaffolds, finding that ER alpha is primarily regulated by the matrix stiffness. Matrix stiffness upregulates the ER alpha signaling via stress-mediated p38 activation and H3K27me3-mediated epigenetic regulation. The finding that the matrix stiffness is a central cue to the ER alpha phenotype reveals a mechanobiological component in breast tissue hormonal signaling and enables the development of novel therapeutic interventions. Subject terms: ER-positive (ER + ), breast cancer, ex vivo model, preclinical model, PDEC, stiffness, p38 SAPK.Reliable luminal estrogen receptor (ER alpha+) breast cancer models are limited. Here, the authors use patient derived breast epithelial and breast cancer explant cultures grown in several extracellular matrix scaffolds and show that ER alpha expression is regulated by matrix stiffness via stress-mediated p38 activation and H3K27me3-mediated epigenetic regulation.</p

Biomimeettinen materiaalisuunnittelu kohti sitkeitä nanokomposiitteja ja myötöjäykistyviä hydrogeelejä

Defence is held on 28.5.2021 11:15 – 15:15. Remote https://aalto.zoom.us/j/61911311409Biological organisms use only a few chemical elements to construct materials, such as proteins, polysaccharides, and minerals, which are efficiently processed into hierarchical structures across length scales. These clever multi-component designs produce materials and structure with remarkably improved mechanical properties and functionalities relative to the original components. The field of biomimetics aims to mimic these well-adapted design strategies, structures and functions to solve material engineering problems. This thesis focuses on two nature-inspired systems: (i) tough and strong nacre-mimetic nanocomposites, and (ii) strain-stiffening biopolymer hydrogels and their application for cell culturing. Both systems consist of nanoscale components and their mechanical properties and structure are studied. The design strategy for tough and strong composites is inspired by the nacre, a biomaterial with outstanding mechanical properties. Here the structural organization of nacre is mimicked via self-assembling core-shell structured colloidal platelets, i.e. nanoclay coated with a polymer, via vacuum filtration. In publications I and II, we alter the interactions between the colloidal platelets with DNA-based monophosphates and demonstrate a simple way to modify intermolecular interactions resulting in increased stiffness, strength, and toughness. Like natural nacre, these nanocomposites are sensitive to humidity. In publication III, we study the effect of water in polymer-clay nanocomposites and find that the glass transition temperature of the nanoconfined polymer is lowered due to residual water. The second part is inspired by typical extracellular matrix-based protein gels, which show strain-stiffening. In publication IV, we show that agarose hydrogels are strain-stiffening, consisting of helically twisted semiflexible fibrillar networks. In publication V, we analyze this strain-stiffening response more closely and simultaneously show, for the first time, that agarose gels also contract when sheared, which is seen as negative normal force and normal stress difference. Our main findings indicate that the mechanical response of agarose networks is enthalpic and that connectivity dictates their strain-stiffening response similarly as in collagen gels. Finally, in publication VI, we present an application for agarose hydrogels as a luminal cell identity and estrogen receptor α+-preserving scaffold for breast cancer tissue explant culture. Base on a biomimetic materials design approach, the first part of this thesis illustrates a simple method to control the mechanical properties of layered clay-polymer nanocomposites. The second part presents insights into the fibril network mechanics of agarose hydrogels. The final publication introduces a reliable agarose-based preclinical model, which can be used as a platform for breast cancer drug development and personalized cancer therapy.Biologisten luonnonmateriaalien, kuten proteiinien, polysakkaridien ja mineraalien, pohjana ovat vain muutamat alkuaineet. Biologisissa organismeissa ne muodostavat sopivia hierarkisia komposiittirakenteita, joiden ominaisuudet ja toiminnot ovat moninkertaisesti paremmat yksittäisiin komponentteihin nähden. Biomimetiikka pyrkii ottamaan mallia näistä luonnossa esiintyvistä materiaaleista, rakenteista ja toiminnoista ja soveltaa niitä teknisten ongelmien ratkaisussa. Väitöskirjassani keskityn kahteen luontoa esikuvanaan käyttävään materiaalisovellukseen: (i) lujat ja sitkeät helmiäistä jäljittelevät nanokomposiitit ja (ii) myötöjäykistyvät 3D-solunkasvatusalustat. Molemmissa sovelluskohteissa muokkasin nanokoon komponenteista koostuvien materiaalien mekaanisia ominaisuuksia ja tarkastelin loppumateriaalien rakenteen ja ominaisuuksien välisiä riippuvuuksia. Simpukan helmiäisen lujaa ja sitkeää rakennetta matkittiin kolloidisilla hiutaleilla, joiden sisus koostui yksittäisistä nanosavihiutaleista ja ulkokuori polymeereista. Kolloidiset hiutaleet itsejärjestyivät kerroksellisiksi nanokomposiittikalvoiksi. Julkaisuissa I ja II muokkasimme kolloidisten hiutaleiden vuorovaikutuksia DNA-pohjaisilla vetysitoutuvilla monofosfaateilla, mikä lisäsi lujuutta, jäykkyyttä ja sitkeyttä. Nanokomposiittikalvot olivat herkkiä kosteudelle. Julkaisussa III tutkimme kosteuden vaikutusta savipolymeerinanokomposiittikalvoihin. Näytimme, että kosteus lisäsi polymeerifaasin dynamiikkaa ja alensi nanokomposiitin lasitransitiolämpötilaa, mikä lisäsi kalvon sitkeyttä. Väitöskirjani jälkimmäisen osan materiaalien taustalla ovat myötöjäykistyvät proteiinigeelit, jotka on valmistettu soluvälianeen tai solun sisäisen tukirangan proteiineista. Julkaisussa IV näytämme, että agaroosihydrogeelit ovat myötöjäykistyviä. Julkaisussa V perehdyimme myötöjäykistymisilmiöön tarkemmin ja osoitimme, että agaroosihydrogeelit puristuvat kokoon leikkausjännityksessä. Tulokset viittaavat siihen, että entalpia määrittää agaroosigeelien mekaanista vastetta ja verkkorakenteen koordinaatioluku määrittelee myötöjäykistymisvasteen, kuten aiemmin on havaittu kollageenigeeleillä. Lopuksi julkaisussa VI näytämme, että agaroosihydrogeeli soveltuu solunkasvatusalustaksi luminaalisille ER-α+ rintasyöville. Biomimeettisen materiaalisuunnittelun tuloksena osoitan väitöskirjassani tavan kontrolloida savipolymeerinanokomposiittien mekaanisia ominaisuuksia. Näytän myös, kuinka agaroosihydrogeelien kuituverkkorakenne säätelee niiden mekaanista käyttäytymistä. Lopuksi esittelen agaroosipohjaisen hydrogeelikasvatusalustan, joka sopii myös mallialustaksi syöpälääketutkimukseen ja yksilöllisiin prekliinisiin rintasyöpälääketestauksiin

Strain Stiffening and Negative Normal Force of Agarose Hydrogels

| openaire: EC/H2020/742829/EU//DRIVENInspired by the specific strain stiffening and negative normal force phenomena in several biological networks, herein, we show strain stiffening and negative normal force in agarose hydrogels. We use both pre-strain and strain amplitude sweep protocols in dynamic rheological measurements where the gel slip was suppressed by the in situ gelation in the cross-hatched parallel plate rheometer geometry. Within the stiffening region, we show the scaling relation for the differential modulus K ∝ σ1, where σ is stress. The strain at the onset of stiffening is almost constant throughout the concentration range. The gels show negative apparent normal stress difference when sheared as a result of the gel contraction. The pore size of the hydrogel is large enough to allow water to move with respect to the network to balance the pressure difference caused by the hoop stress. The rheological analysis together with scanning electron microscopy suggests that the agarose gels can be described using subisostatic athermal network models where the connectivity dictates the stiffening behavior. Therefore, the simple agarose gels appear to capture several of the viscoelastic properties, which were previously thought to be characteristic to biological protein macromolecules.Peer reviewe

Deoxyguanosine Phosphate Mediated Sacrificial Bonds Promote Synergistic Mechanical Properties in Nacre-Mimetic Nanocomposites

We show that functionalizing polymer-coated colloidal nanoplatelets with guanosine groups allows synergistic increase of mechanical properties in nacre-mimetic lamellar self-assemblies. Anionic montmorillonite (MTM) was first coated using cationic poly(diallyldimethylammonium chloride) (PDADMAC) to prepare core–shell colloidal platelets, and subsequently the remaining chloride counterions allowed exchange to functional anionic 2′-deoxyguanosine 5′-monophosphate (dGMP) counterions, containing hydrogen bonding donors and acceptors. The compositions were studied using elemental analysis, scanning and transmission electron microscopy, wide-angle X-ray scattering, and tensile testing. The lamellar spacing between the clays increases from 1.85 to 2.14 nm upon addition of the dGMP. Adding dGMP increases the elastic modulus, tensile strength, and strain 33.0%, 40.9%, and 5.6%, respectively, to 13.5 GPa, 67 MPa, and 1.24%, at 50% relative humidity. This leads to an improved toughness seen as a ca. 50% increase of the work-to-failure. This is noteworthy, as previously it has been observed that connecting the core–shell nanoclay platelets covalently or ionically leads to increase of the stiffness but to reduced strain. We suggest that the dynamic supramolecular bonds allow slippage and sacrificial bonds between the self-assembling nanoplatelets, thus promoting toughness, still providing dynamic interactions between the platelets.Peer reviewe

Hydration and dynamic state of nanoconfined polymer layers govern toughness in nacre-mimetic nanocomposites

Biological high-performance composites inspire to create new tough, strong, and stiff structural materials. We show a brittle-to-ductile transition in a self-assembled nacre-inspired poly(vinyl alcohol)/nanoclay composite based on a hydration-induced glass-to-rubber transition in the 2D-nanoconfined poly(vinyl alcohol) layers. The findings open routes to design dissipative toughening mechanisms to combine stiffness and strength in nanocomposites.Funded by: Graduate School for Materials Physics, Emil Aaltonen Foundation, and Academy of Finland.Peer Reviewe

Deoxyguanosine Phosphate Mediated Sacrificial Bonds Promote Synergistic Mechanical Properties in Nacre-Mimetic Nanocomposites

We show that functionalizing polymer-coated colloidal nanoplatelets with guanosine groups allows synergistic increase of mechanical properties in nacre-mimetic lamellar self-assemblies. Anionic montmorillonite (MTM) was first coated using cationic poly­(diallyldimethylammonium chloride) (PDADMAC) to prepare core–shell colloidal platelets, and subsequently the remaining chloride counterions allowed exchange to functional anionic 2′-deoxyguanosine 5′-monophosphate (dGMP) counterions, containing hydrogen bonding donors and acceptors. The compositions were studied using elemental analysis, scanning and transmission electron microscopy, wide-angle X-ray scattering, and tensile testing. The lamellar spacing between the clays increases from 1.85 to 2.14 nm upon addition of the dGMP. Adding dGMP increases the elastic modulus, tensile strength, and strain 33.0%, 40.9%, and 5.6%, respectively, to 13.5 GPa, 67 MPa, and 1.24%, at 50% relative humidity. This leads to an improved toughness seen as a ca. 50% increase of the work-to-failure. This is noteworthy, as previously it has been observed that connecting the core–shell nanoclay platelets covalently or ionically leads to increase of the stiffness but to reduced strain. We suggest that the dynamic supramolecular bonds allow slippage and sacrificial bonds between the self-assembling nanoplatelets, thus promoting toughness, still providing dynamic interactions between the platelets